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Single-step selection of mammalian cell mutants deficient in CTP synthetase
1Department of Medicine, Medical College, National Cheng Kung University, Tainan, Taiwan.
Abstract:
A single-step selection of Chinese hamster V79 cells deficient in CTP synthetase (CTPS-) is presented. The underlying principle of the direct selection is the differential and efficient killing of synchronized wild-type cells through incorporation of [3H]uridine and [3H]thymidine. The CTPS- mutant cells were recovered by virtue of their not engaging in DNA synthesis, because (1) CTPS- cells are deficient in CTP synthetase and thus are unable to convert [3H]UTP into [3H]CTP, which eventually is converted into [3H]dCTP and incorporated into DNA; (2) the growth of CTPS- mutant cells was arrested as a result of cytidine deprivation, thus escaping the killing by the incorporation of [3H]thymidine. The isolated mutant clones are auxotrophic for cytidine and are stable in phenotype with a reversion frequency of less than 1 x 10(-7). The mutant cells have no or very low CTP synthetase activity when tested by in vitro CTP synthetase assay or by whole-cell [3H]uridine labeling assay. This modified "tritium suicide" method combined with the S-phase cell synchronization could provide a powerful means for the recovery from the cell population of nondividing mutant cells that are auxotrophic for some special nutrient requirement.
Insights
This study presents a novel method for isolating Chinese hamster V79 cells lacking CTP synthetase (CTPS-). The technique utilizes a modified "tritium suicide" approach combined with cell synchronization to efficiently select for auxotrophic mutant cells.
Area of Science:
- Cell Biology
- Genetics
- Biochemistry
Background:
- CTP synthetase (CTPS) is crucial for nucleotide biosynthesis.
- Isolating specific auxotrophic mutants can be challenging.
- Previous methods for mutant selection were less efficient.
Purpose of the Study:
- To develop a single-step selection method for Chinese hamster V79 cells deficient in CTP synthetase (CTPS-).
- To efficiently recover non-dividing mutant cells with specific nutrient requirements.
Main Methods:
- Employed a modified "tritium suicide" technique.
- Utilized synchronized wild-type cells and CTPS- mutant cells.
- Incorporated [3H]uridine and [3H]thymidine for differential cell killing.
Main Results:
- Successfully isolated stable CTPS- mutant clones with a reversion frequency below 1 x 10(-7).
- CTPS- mutant cells showed no or very low CTP synthetase activity.
- The method leverages cytidine deprivation and inability to incorporate radiolabeled nucleotides into DNA.
Conclusions:
- The developed method is a powerful tool for isolating auxotrophic mutant cells.
- Combines cell synchronization with a modified tritium suicide assay for efficient selection.
- Applicable for recovering non-dividing mutant cells with specific nutrient dependencies.